In scanning microscopy, a sample is illuminated with a light beam in order to observe the detection light emitted, as reflected or fluorescent light, from the sample. The focus of an illuminating light beam is moved in a sample plane using a controllable beam deflection device, generally by tilting two mirrors; the deflection axes usually being perpendicular to one another so that one mirror deflects in the x-direction and the other in the y-direction. Tilting of the mirrors is brought about, for example, by galvanometer positioning elements. The power of the detection light coming from the sample is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to determine the current mirror position. In confocal scanning microscopy specifically, a sample is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally includes a light source, a focusing optical system used to focus the light of the source onto a pinhole (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detection or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the sample travels back via the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. This detection arrangement is called a “descan” arrangement. Detection light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that point information is obtained which, by sequential scanning of the sample with the focus of the illuminating light beam, results in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers.
United States Patent Application US 2002/0097489 A1 describes a microscope providing evanescent wave illumination of a sample. The microscope contains a white light source, whose light passes through a slit aperture and the microscope objective and is coupled into the sample-carrying slide to provide evanescent wave illumination. The illuminating light propagates in the slide by total internal reflection, the illumination of the sample occurring only in the area of the evanescent field protruding from the slide. Microscopes of this type are known by the term TIRFM (Total Internal Reflection Fluorescent Microscope).
The z-resolution of TIRF microscopes is extremely good because the evanescent field extends only about 100 nm into the sample.